‘In-Format’ screening of a novel bispecific antibody format reveals significant potency improvements relative to unformatted molecules

Abstract

Bispecific antibodies (BsAbs) are emerging as an important class of biopharmaceutical. The majority of BsAbs are created from conventional antibodies or fragments engineered into more complex configurations. A recurring challenge in their development, however, is the identification of components that are optimised for inclusion in the final format in order to deliver both efficacy and robust biophysical properties. Using a modular BsAb format, the mAb-dAb, we assessed whether an 'in-format' screening approach, designed to select format-compatible domain antibodies, could expedite lead discovery. Human nerve growth factor (NGF) was selected as an antigen to validate the approach; domain antibody (dAb) libraries were screened, panels of binders identified, and binding affinities and potencies compared for selected dAbs and corresponding mAb-dAbs. A number of dAbs that exhibited high potency (IC 50 ) when assessed in-format were identified. In contrast, the corresponding dAb monomers had »1000-fold lower potency than the formatted dAbs; such dAb monomers would therefore have been omitted from further characterization. Subsequent stoichiometric analyses of mAb-dAbs bound to NGF, or an additional target antigen (vascular endothelial growth factor), suggested different target binding modes; this indicates that the observed potency improvements cannot be attributed simply to an avidity effect offered by the mAb-dAb format. We conclude that, for certain antigens, screening na€ ıve selection outputs directly in-format enables the identification of a subset of format-compatible dAbs, and that this offers substantial benefits in terms of molecular properties and development time.

CTLA-4 and PD-1 dual blockade induces SIV reactivation without control of rebound after antiretroviral therapy interruption

“…The IgG1 anti-CTLA-4 mAb (αCTLA-4) is a fully human, biosimilar formulation of ipilimumab (Bristol-Myers Squibb) that retains Fcγ binding activity. The IgG1 bispecific αCTLA-4/PD-1 mAbdAb (BsAb) has a human variable and rhesus constant region with a LAGA mutationdisabled Fc receptor, and was constructed with a flexible fibronectin linker connecting the C terminus of the mAb heavy chain to a PD-1 domain antibody (dAb) 42 . The antagonistic anti-PD-1 dAb was identified through phage display, and bound PD-1-expressing cells with a half-maximum effective concentration of 2 nm, thereby efficiently blocking interactions with PDL-1/PDL-2 43 .…”

Intein mediated high throughput screening for bispecific antibodies

“…[6,15,16] BsAb development is usually time-and labor-intensive, involves higher manufacturing costs, and requires safety and efficacy validation of each mAb and in combination. [17] Approaches used to identify the single binding moieties of an envisioned bsAb tend to focus on reduction of individual clone numbers by filters such as affinity, target specificity, optimally domain or epitope mapping. Most intended modes of action are not conveyed by a single binding moiety, but through the combination to be identified, for example, selective effector cell recruitment, target-specific Fc mediated effector functions or enhanced selectivity binding by avidity or even overall modes of action that are not predictable.…”

Toward Drug-Like Multispecific Antibodies by Design

“…Of course, this type of assay relies on the viability of the display system following heat shock, which has led to the development of a new thermostable yeast strain for use in such heat shock screens [ 47 ]. Although this fragment-based approach has proven effective, recent advances in screening intact bispecific antibodies suggests that the final format of an antibody can have unexpected consequences for potency, and may therefore identify variants with optimal properties that may be missed by the fragment-based approaches [ 48 ].…”